Endothelium-Derived Relaxing Factor
Endothelial cells have been shown to produce a potent vasodilator known as Endothelium-Derived Relaxing Factor (EDRF). Many naturally occurring substances which act as physiological vasodilators mediate all or part of their action by stimulating the release of EDRF. Examples of such substances include acetylcholine, histamine, bradykinin, leukotrienes, ADP, and ATP. Recent studies have identified EDRF as nitric oxide, a short lived, unstable compound (Ignarro et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:9265-9269 and Palmer et al., 1987, Nature 327:524-526).
L-Arginine is the metabolic precursor of EDRF (Schmidt et al., 1988, Eur. J. Pharmacol. 154:213-216). N.sup.G -methyl-L-arginine is a competitive inhibitor of the biosynthetic pathway of EDRF (Palmer et al., 1988, Nature 333:664-666). Administration of N.sup.G -methyl-L-arginine to guinea pigs and rabbits has been shown to increase blood pressure (Aisaka et al., 1989, Biochem. Biophys. Res. Commun. 160:881-886). Nitric oxide (NO) appears to be synthesized from L-arginine by the enzyme, NO synthase; the coproduct is L-citrulline (Moncada et al., 1991, J. Cardiovascular Pharmacol. 17 (Suppl. 3):S1-S9). NO is an endogenous stimulator for soluble guanylate cyclase.
Nitric oxide has been found to be produced by macrophages, endothelial cells, neutrophils, Kupffer cells and hepatocytes, murine fibroblasts stimulated with cytokines, and EMT-6 cells, a spontaneous murine mammary adenocarcinoma cell line when treated with cytokine (reviewed in Moncada et al., 1991, Pharmacol. Reviews 43:109-142). Specifically, macrophage cells become activated by 12-36 hour treatments with gamma-interferon, bacterial endotoxin and various cytokines (reviewed in Collier and Vallance, 1989, Trends in Pharmacol. Sci. 10:427-431).
Endothelial cells in the presence of gamma-interferon, have been found to secrete large quantities of arginine-derived nitrogen oxides after activation by tumor necrosis factor (TNF) or endotoxin (Kilbourn and Belloni, 1990, J. Natl. Cancer Inst. 82:772-776). TNF causes marked hypotension in mammals (Tracey et al., 1986, Gynecol. Obstet. 164:415-422; Old, 1985, Science 230:630-632). Additionally, TNF is thought to mediate the vascular collapse resulting from bacterial endotoxin (Beutler et al., 1985, Science 229:869-871). It has recently been shown that arginine derivatives inhibit systemic hypotension associated with nitric oxide production, specifically treatment with TNF, gamma interferon, interleukin-2, and bacterial endotoxin (Kilbourn et al., U.S. Pat. No. 5,028,627, issued Jul. 2, 1991; PCT Application no. WO 91/04023, published Apr. 4, 1991; and Kilbourn et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:3629-3632). Nitric oxide overproduction is also thought to be involved in numerous other pathogenic or potentially pathogenic syndromes. For example, some of these syndromes are thought to be associated with malaria, senescence and diabetes. The procedures of the present invention may also be used to prevent, inhibit and/or alleviate such NO-related syndromes.
Interaction of Hemoglobin with Endothelium-Derived Relaxation Factor
Nitric oxide reacts with hemoglobin to form nitrosylhemoglobin (Kosaka et al., 1989, Free Radical Biology 7:653-658). Nitrosylhemoglobin reacts with oxygen to yield nitrate and methemoglobin, which is rapidly reduced by methemoglobin reductase. At least part of the nitric oxide is oxidized by oxygen to NO.sub.2, which is in turn converted to nitrite and nitrate.
The formation of nitrosylhemoglobin has been used to quantify nitric oxide present in mice given bacterial endotoxin, specifically by using electron paramagnetic resonance (EPR) spectroscopy to detect NO liganded to hemoglobin (Wang et al., 1991, Life Sciences 49:55-60). Although bacterial endotoxin induces septic shock, hypotension was not observed in the Wang et al. study. Those skilled in the art recognize that such EPR studies may be used to quantitate the binding of NO to other hemoproteins as well.
Hemoglobin has also been found to inhibit nonvascular relaxant responses to EDRF (Buga et al., 1989, Eur. J. Pharmacol. 161:61-72). Furthermore, hemoglobin at 1 .mu.M reduced and at 10 .mu.M abolished the endothelium-dependent relaxation induced by acetylcholine or by A23187 in rabbit aortic rings (Martin et al., 1985, J. Pharmacol. Exp. Ther. 232:708-716). It was hypothesized by Martin et al. that the hemoglobin inhibits endothelium-dependent induced relaxation by binding nitric oxide.
Septic Shock
Septic shock is characterized by inadequate tissue perfusion and is usually caused by gram-negative enteric bacilli such as Escherichia coli, Klebsiella, Enterobacter, Pseudomonas, Serratia, and Bacteroides (see Harrison's INTERNAL MEDICINE, 10th Ed., vol. 1, Petersdorf et al., eds., McGraw Hill, N.Y. 1983). Gram-negative bacilli possess endotoxin, also known as lipopolysaccharide (LPS), which is a cell-wall component that can activate leukocytes in minute amounts in the blood.
Septic shock is characterized by chills, fever, nausea, vomiting, diarrhea, and prostration. The subsequent development of septic shock is characterized by tachycardia, tachypnea, hypotension, peripheral cyanosis, mental obtundation, and oliguria. As shock progresses, oliguria persists, and heart failure, respiratory insufficiency, and coma supervene. Death usually results from pulmonary edema, generalized anoxemia secondary to respiratory insufficiency, cardiac arrhythmia, disseminated intravascular coagulation with bleeding, cerebral anoxia, or a combination of the above.
Most of the damage caused by septic shock is thought to be caused by endotoxin. It has also been hypothesized that nitric oxide plays a major role in effecting hypotension in those exposed to endotoxin (Kilbourn et al. Biochem. and Biophys. Res. Comm. 1990, vol. 172:1132-1138). Studies have shown that the hypotension and loss of vascular responsiveness resulting from endotoxin administration is reversed by the administration of analogues of L-arginine which inhibit nitric oxide production (Parratt and Stoclet, 1991, Applied Cardiopulmonary Pathophysiology 4:143-149; and Kilbourn et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:3629-3632).
In certain cases, acute blood loss may cause hypotension. After initial recovery due to standard hypotension treatment, a breach of intestinal integrity may subsequently cause septic hypotension. Such a syndrome is readily treated according to the methods of the present invention.
Treatments for Septic Shock
There are currently a number of clinical procedures available for the treatment of septic shock (reviewed in Harrison's INTERNAL MEDICINE, 10th Ed., vol.l, Petersdorf et al., eds., McGraw Hill, N.Y. 1983). However each has its drawbacks. One treatment involves the replacement of blood volume with blood, plasma, or other fluids such as human serum albumin in appropriate electrolyte solutions such as dextrose saline and bicarbonate. However, large quantities of these substances are required and may amount to 8 to 12 liters in only a few hours, leading to massive shifts in fluid and electrolyte balances.
Antibiotics may also be used to treat septic shock. However, to determine the appropriate antibiotics to use, blood cultures must be taken and evaluated. Such cultures take time. Additionally, antibiotics may initially worsen shock caused by bacteria since they kill the bacteria and the dying organisms can release even more lipopolysaccharide into the body.
Vasoactive drugs such as beta-receptor stimulants (e.g. isoproterenol and dopamine) and alpha-receptor blocking agents (phenoxybenzamine and phentolamine) have also been therapeutically used since septic shock is accompanied by maximal stimulation of alpha-adrenergic receptors. However, all of these drugs may have serious side effects. In addition, studies by Stoclet et al. (Amer. J. Physiol. 259:H1038-1043) suggest that endotoxic shock and NO production can cause lowered sensitivity to these agents, decreasing their effectiveness. The iron hemoprotein therapy of the present invention may be used to reverse this loss of sensitivity.
Another treatment approach involves using human antiserum to E. coli J5, a mutant strain that produces endotoxin without the variable oligosaccharide side chains. However, since such antiserum comes from many different human donors with varying immune responses to the antigen, it cannot be rigidly standardized for effectiveness. Such antiserum may also transmit infectious agents. To circumvent these problems, monoclonal antibodies that bind to endotoxin are currently being tested in patients suffering from septic shock (reviewed in Johnston, 1991, J. NIH Res. 3:61-65). A disadvantage of this approach is that treatment of septic patients with antiendotoxin antibodies has no immediate effect on blood pressure and may not work at all if patients are not treated at an early stage.
Another approach used to treat septic shock involves administering bactericidal permeability-increasing protein (BPI), a human protein derived from neutrophil granules. This protein binds to LPS. Versions of the molecule are currently being developed (reviewed in Johnston, 1991, J. NIH Res. 3:61-65).
Approaches are also being developed for treating septic shock by preventing endotoxin from activating leukocytes that start the inflammatory response (reviewed in Johnston, 1991, J. NIH Res. 3:61-65). Examples of such approaches include administering a soluble interleukin-1 (IL-1) receptor, an IL-1 receptor antagonist, a monoclonal antibody to TNF-.alpha., soluble TNF receptor and a monoclonal antibody to TNF-.alpha. receptor. All of these approaches suffer from the fact that blockade of the cytokine receptor interaction does not have an immediate effect on nitric oxide production by NO synthase (i.e.--once induced by cytokines, NO synthase continues to produce NO even after removal of the stimulus).
The present invention involves the treatment of a nitric oxide-induced malady such as hypotension by administration of a metal-containing hemoprotein which has substantial affinity for nitric oxide and/or catalyzes nitric oxide oxidation to nitrates or nitrites. Iron is a preferred metal, although other transition metals such as nickel, ruthenium, cobalt or copper may be bound to heme and be nitric oxide-reactive. One preferred hemoprotein is hemoglobin, particularly recombinant hemoglobin. Another preferred hemoprotein is hemalbumin. Myoglobin, which also is known to bind nitric oxide, may also be utilized. Additional iron hemoproteins, including peroxidase, catalase, cytochrome oxidase and certain cytochromes, such as cytochrome P-450, are known and may be used to demonstrate the above mentioned nitric oxide-related effects. Particularly preferred hemoglobins are those which have a long circulating lifetime, e.g., those which have a poor affinity for haptoglobin. Those skilled in the art will recognize that other iron heme-containing proteins or peptides binding NO may be administered to an animal in a pharmaceutically effective amount to remove nitric oxide and prevent or treat hypotension or other NO-induced deleterious effects.
Despite previous results showing the interaction of iron hemoproteins such as hemoglobin with nitric oxide, the therapeutically effective results of the present inventive method are quite surprising and unexpected. Such a result could not have been expected in view of the fact that normal mammalian blood fluid is replete with hemoglobin contained in red blood cells which contain about 34% hemoglobin. Such cells usually constitute over 43% of the blood. Thus blood is about 15% hemoglobin. Additionally, nitric oxide is quite lipophilic and readily penetrates red blood cells. One skilled in the art would have expected that any effects of a free iron hemoprotein such as hemoglobin in the blood to remove deleterious nitric oxide would have been insignificant as compared to the effects of preexisting hemoglobin contained in red blood cells. The basis for the surprising and unexpectedly effective results of the present invention are incompletely understood at the present time, but, as the present invention is described, it will be clear to those of skill in the art that the free hemoproteins of the present invention such as hemoprotein are phenomenally effective for therapeutic purposes related to a reversal of deleterious physiological effects induced by nitric oxide in vivo. Hemalbumin, not binding haptoglobin and having very favorable in vivo circulatory characteristics, is particularly valuable.